Any discussion of the background art throughout the specification should in no way be considered as an admission that such background art is prior art, nor that such background art is widely known or forms part of the common general knowledge in the field.
Liver fibrosis is a harmful result from a majority of chronic damages to the liver. It is often featured by an abnormal increase of collagen in the extracellular matrix (ECM). Quantitative characterization of collagen in intact tissue structure is therefore essential for understanding and controlling this harmful process.
More generally, liver fibrosis is characterized by increased deposition of ECM, such as fibrillar collagens type I and III, leading to the derangement of liver architecture, portal hypertension, and the development of esophageal varices, edema and ascites. In addition to these changes in morphology, the presence of excess collagen fibers around the hepatocytes impairs their ability to receive enough nutrition, and the accumulation in sinusoids obstructs blood flow, resulting in unhealthy hepatocytes and great decrease in liver function.
Assessing the collagen fibres accurately and dynamically is therefore important for healthcare researchers to monitor the progression, understand the mechanism, and evaluate the therapy strategy.
The classical methods are histological stains (e.g. Masson's trichrome stain, dsmin and vimentin stain etc) and biochemical analysis (e.g. serum laminin analysis). However, traditional histological staining methods limit the assessment to thin tissue slice due to limitations on dye diffusion and the optical penetration of the imaging techniques, and the biochemical analysis loses the spatial distribution information, thereby making it prohibitive to image dynamically in three dimensions (3D).
Furthermore, knowledge of cell morphology information is also important to predict the collagen deposition and distribution during the progression of fibrosis. However, the cell morphology is prone to disintegration during histological staining processes. In addition, for tissue culture work, it is necessary to have enough cell layers in order to keep its structure and functions, and avoid influence on cell behaviour from fluorescence dyes and exogenous proteins for labelling.
All the limitations outlined above make the usual histological staining processes far from ideal. Therefore, it is highly demanded to develop an imaging technique that can accommodate thick tissue slice, in which no staining is required. With the development of mode-lock laser techniques, nonlinear optical microscopy (multi-photon excitation fluorescence and multi-harmonic generation) has been proven to be a promising solution for thick tissue imaging and opens up a realm of thick tissue imaging with a spatial resolution at micrometer scale. The unique advantages of nonlinear optical microscopy include deeper penetration depth, better spatial resolution, intrinsic optical sectioning, and less photo-bleaching and phototoxicity. Besides these general merits, second harmonic generation (SHG) microscopy, in which two photons are converted into one with double energy and no excited state is involved with a finite lifetime, is sensitive to the alignment, orientation, polarization, and local symmetry of chiral molecules. Therefore SHG can convey rich structural information as well as chemical properties.
Interestingly, some highly-ordered intrinsic structures in cells and tissues, e.g. microtubule, collagen, and myosin, can generate strong SHG signals. By detecting the SHG signals, there is no need to label these structures with exogenous dyes or fluorescent proteins any more, offering a non-invasive methodology to monitor the dynamical changes of tissues in intact state. Due to these unparalleled characteristics, SHG microscopy has attracted extensive attentions in life science and applications have been reported in many varied fields such as in surface property studies, transmembrane potential measurements, and cell imaging to name but a few.
In the tissue imaging realm, SHG images of rat tail tendon were first reported in 1986 and subsequently SHG imaging of other tissues, such as fish scale, skin, the cornea, brain tissue, muscle tissue and tumors have been studied. Among the endogenous proteins, collagen type I was investigated using SHG most thoroughly because of its larger second order susceptibility and well defined structure. To increase the understanding of effects of laser parameters and interpretation of the images obtained, the fundamental principles governing SHG from collagen have also been explored.
However, despite the extensive activity in tissue imaging, nonlinear optical imaging for liver tissue is rarely reported. The main difficulties with nonlinear imaging for liver tissue stem from the fact that liver tissue is a highly light scattering material and there is less fibrillar collagen in normal condition, resulting in shallow optical penetration and weak SHG signals.
Nonlinear optical imaging has been used for analysis of liver tissue by Cox et al. [G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, 3-Dimensional imaging of collagen using second harmonic generation, J. Struct. Biol. 141, 53-62, 2003], however, the reported images were from cirrhotic liver slices (50 μm in thickness), in which much more collagen than fibrosis had been generated.
In order to understand the progression of fibrosis and achieve better prognosis in clinical practices, it is necessary to detect fibrosis from early stage, which requires higher detection sensitivity and better resolution.